Article pubs.acs.org/JAFC
Metabolism of the Insecticide Metofluthrin in Cabbage (Brassica oleracea) Daisuke Ando,* Masao Fukushima, Takuo Fujisawa, and Toshiyuki Katagi Environmental Health Science Laboratory, Sumitomo Chemical Company, Ltd., 4-2-1 Takatsukasa, Takarazuka, Hyogo 665-8555, Japan ABSTRACT: The metabolic fate of metofluthrin [2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl (E,Z)-(1R,3R)-2,2-dimethyl-3(prop-1-enyl)cyclopropanecarboxylate] separately labeled with 14C at the carbonyl carbon and the α-position of the 4methoxymethylbenzyl ring was studied in cabbage (Brassica oleracea). An acetonitrile solution of 14C-metofluthrin at 431 g ai ha−1 was once applied topically to cabbage leaves at head-forming stage, and the plants were grown for up to 14 days. Each isomer of metofluthrin applied onto the leaf surface rapidly volatilized into the air and was scarcely translocated to the untreated portion. On the leaf surface, metofluthrin was primarily degraded through ozonolysis of the propenyl side chain to produce the secondary ozonide, which further decomposed to the corresponding aldehyde and carboxylic acid derivatives. In the leaf tissues, the 1R-trans-Z isomer was mainly metabolized to its dihydrodiol derivative probably via an epoxy intermediate followed by saccharide conjugation in parallel with the ester cleavage, whereas no specific metabolite was dominant for the 1R-trans-E isomer. Isomerization of metofluthrin at the cyclopropyl ring was negligible for both isomers. In this study, the chemical structure of each secondary ozonide derivative was fully elucidated by the various modes of liquid chromatography−mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy together with cochromatography with the synthetic standard, and their cis/trans configuration was examined by the nuclear Overhauser effect (NOE) difference NMR spectrum. KEYWORDS: metofluthrin, plant metabolism, ozonide, diastereomer
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INTRODUCTION Metofluthrin (1) [SumiOne, Eminence; 2,3,5,6-tetrafluoro-4(methoxymethyl)benzyl (E,Z)-(1R,3R)-2,2-dimethyl-3-(prop1-enyl)cyclopropanecarboxylate] is a pyrethroid insecticide for household and public hygiene usages developed by Sumitomo Chemical Co., Ltd., and has a strong knockdown activity, especially against mosquitoes. Because of its high volatility (1.96 × 10−3 Pa) with a low mammalian toxicity, 1 can be used in nonheated formulations such as fan-type, paper, and resin emanators.1 Incidentally, the presence of the two optical centers at the cyclopropenyl ring and one geometrical isomerism at the propenyl side chain result in eight isomers, and 1 consists of the biologically active 1R-trans isomers having an E/Z geometrical ratio of 1/8, abbreviated as 1-RTE and 1RTZ.1 The extremely limited emission of 1 to the environment is most likely because it is mainly used indoors. However, as there is no geographic restriction of its use, that is, personal outside insect repellent, 1 may directly or indirectly reach a nontarget environment such as a water body, soil, and plants. In addition, with regard to biocide, submission of relevant data to the European Union and the U.S. Environmental Protection Agency regarding environmental fate has recently become inevitable, irrespective of the use pattern especially due to the possible contamination by sewage treatment plant effluent. From these aspects, it is important to obtain experimental data on 1 to show that it is benign to the environment. The aerobic soil metabolism in two U.S. soils has shown that 1-RTZ and 1RTE rapidly degrade at similar half-lives of 2.3−3.5 days via cleavage of the ester linkage to produce the corresponding alcohol (10) and acid (7), followed by successive oxidation at © 2011 American Chemical Society
the prop-1-enyl group and the benzyl carbon to form dicarboxylic acid (11) and terephthalic acid (9) derivatives, respectively.2 Further degradation of the metabolites resulted in production of carbon dioxide, but little enantiomerization and geometrical isomerization proceeded. Radioactivity was also detected as the soil bound residues. The soil adsorption coefficient (Koc) of 1-RTZ in three German soils was determined to be 3553−6142 mL g−1 oc by the batch equilibrium method.2 Under illumination on the moisturecontrolled soil, 1-RTZ degraded with a half-life of 8.1−12.0 days.3,4 The photodegradation pathway was oxidation of the double bond at the prop-1-enyl moiety and cleavage of the ester linkage, followed by further decomposition to polar compounds and mineralization to carbon dioxide. The major degradates detected were carbonaldehyde (4) and carboxylic acid (5) derivatives, which were decomposed from ozonide (2) and diol (6) produced by the activated oxygen species, that is, ozone and hydrogen peroxide, respectively. The photoisomerization hardly proceeded throughout the test duration.3 In the hydrolysis study, 1-RTZ was moderately degraded by ester cleavage with a half-life of 26.8 days at pH 9 and 25 °C, whereas it was stable at pH 5 and 7. For aqueous photolysis using a xenon arc lamp, 1-RTZ and 1-RTE rapidly dissipated with halflives of 1.1−3.4 days at pH 7 mainly due to ester cleavage to produce 7 and 10 and oxidation of the olefinic double bond to form 3 and 4, whereas isomerization was a minor route.4 Received: Revised: Accepted: Published: 2607
September 26, 2011 December 21, 2011 December 26, 2011 December 26, 2011 dx.doi.org/10.1021/jf203903r | J. Agric. Food Chem. 2012, 60, 2607−2616
Journal of Agricultural and Food Chemistry
Article
Figure 1. Proposed metabolic pathway of metofluthrin in/on cabbage plant. thalic acid (9); (1R,3R)-2,2-dimethyl-3-(E,Z)-propenylcyclopropanecarboxylic acid (10); (1R,3R)-2,2-dimethyl-3-carboxycyclopropanecarboxylic acid (11). The chemical purity of each standard was determined to be >95% by high-performance liquid chromatography (HPLC). Degradate 2 was prepared according to the following method. Three milligrams of either 1-RTZ or 1-RTE was dissolved in 1 mL of n-hexane solution and cooled to < −40 °C using dry ice/ acetone prior to the ozone oxidation. Ozone gas produced at an approximate concentration of 20 g N−1m−3 using an ozone generator (type 0N-1-2, Nippon Ozone Co., Ltd., Japan) was gently bubbled into the reaction mixture for 3 min at < −40 °C, which resulted in the formation of a white precipitate of crude 2. After the solution returned to room temperature, 2 was successively purified by HPLC, and the solvent was removed using an evaporator and dried in vacuo to obtain a colorless liquid, the chemical purity of which was determined to be >95%. The chemical structure of 2 was confirmed by various modes of NMR and LC-ESI-MS spectroscopies. 2 was stable in acetonitrile, nhexane, and chloroform for a few months in a freezer below 0 °C, but rapidly decomposed to 4 and 5 in aqueous organic media. The following 14C-labeled isomers of 1 were synthesized in our laboratory with the reported methods;2−4 1-RTZ separately labeled at the αposition of 2,3,5,6-tetrafluoro-4-methoxymethylbenzyl ring (benzyl-14C) or the carbonyl carbon (carbonyl-14C) and 1-RTE labled at the carbonyl carbon (carbonyl-14C) with specific activities of 0.1654, 0.1651, and 0.1651 mCi mg−1, respectively. β-Glucosidase (almond) and cellulase (Aspergillus niger) were purchased from Wako Pure Chemical Industries, Ltd. All of the reagents and solvents used were of the analytical grade.
Up to this date, the plant metabolism study of 1, for which information is necessary for the ecotoxicological assessments of nontarget species, is not available. From this standpoint, we have studied the metabolism of 1 in cabbage grown in a greenhouse, which was selected as a model plant because its broad and waxy leaves5 are considered to be suitable to trap and prevent the loss of 1 by rapid volatilization.6 The metabolites formed in/on the plant were clarified by extensive spectrometric analysis using LC-MS and NMR spectroscopy in conjunction with direct chromatographic comparison with the synthetic standards.
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MATERIALS AND METHODS
Chemicals. The nonradiolabeled metofluthrin (1) and its potential degradates as follows were synthesized in our laboratory according to the reported methods.2−4 The structures of the compounds are referred to Figure 1; 2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl (1R,3R)-2,2-dimethyl-3-(3-methyl-1,2,4-trioxolane-5-cyclopropanecarboxylate (2); 2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl (1R,3R)2,2-dimethyl-3-(3-methyl-1,2-epoxy)cyclopropanecarboxylate (3); 2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl (1R,3R)-2,2-dimethyl-3formylcyclopropanecarboxylate (4); 2,3,5,6-tetrafluoro-4(methoxymethyl)benzyl (1R,3R)-2,2-dimethyl-3-carboxycyclopropanecarboxylate (5); 2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl (1R,3R)-2,2-dimethyl-3-(1,2-propanediol)cyclopropanecarboxylate (6); 2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl alcohol (7); 2,3,5,6tetrafluoro-4-(methoxymethyl)benzoic acid (8); tetrafluorotereph2608
dx.doi.org/10.1021/jf203903r | J. Agric. Food Chem. 2012, 60, 2607−2616
Journal of Agricultural and Food Chemistry
Article
subtracted from the dpm value of a measured sample. The 14C in the extracted residues and untreated plant portions was converged and measured as 14CO2 using a Packard model 307 sample oxidizer. 14CO2 produced was absorbed into 9 mL of Packard Carb-CO2 absorber and mixed with 15 mL of Packard Permafluor scintillator, and the radioactivity therein was quantified by LSC. The efficiency of combustion was determined to be >95.8%. Plant Material and Treatment. Cabbage (Brassica oleracea var. capitata, cv. Green Ball) was grown in a 1/5000-are Wagner pot filled with Kasai soil (Hyogo, Japan) in a greenhouse at 25 °C during the day and at 20 °C during the night. The application of each [14C] -1 to the cabbage plants was conducted at the plant growth stage of BBCH 41.7 The average surface area and weight of a cabbage leaf at the application stage were 176.6 cm2 and 10.31 g, respectively. The characterization of Kasai soil is as described as follows: soil texture (%), sand 82.9, silt 8.9, clay 8.2; soil classification, sandy loam; organic carbon content (w/w), 1.7; pH (H2O), 6.6; maximum water-holding capacity (g per 100 g of dry soil), 28.19. The application dose of 1 to cabbage plants was 431 g ai ha−1, which was determined by assuming that a typical commercial can was directly sprayed onto cabbage leaves as the worst-case scenario: 4 g of aerosol of the water-based formulation containing 0.1% (w/w) of 1 was sprayed once onto a square-foot field for the purpose of general insect repellent. Although the isomeric ratio of 1-RTZ and 1-RTE in the active ingredient is 8/1, the same dosing rate was used for each isomer in this study. For the leaf treatment, the dosing solution per leaf was prepared by mixing 0.167 MBq of each [14C]-1 isomer with 0.761 mg of the corresponding unlabeled material in 100 μL of acetonitrile (0.212 MBq mg−1). The prepared dosing solution was topically applied onto nine leaves (three leaves × three pots) using a microsyringe for each label. With respect to the soil treatment, the dosing solution per pot was prepared by combining 1.667 MBq of each [14C]-1 with 0.862 mg of the unlabeled material in 1 mL of acetonitrile and was applied onto 120 g of Kasai soil in a plastic bag using a pipet and thoroughly mixed for 30 min. After evaporation of acetonitrile, it was gently put onto the soil surface of the Wagner pot in which cabbage plants were grown. Sampling, Extraction, and Analysis. For leaf treatment, three cabbage leaves per pot were harvested at 2, 7, and 14 days after treatment. The leaves were individually cut from the stem using scissors, and untreated leaves and stems were similarly sampled with another uncontaminated one. For soil treatment, both cabbage plant and soil were separately sampled at 14 days after treatment. The whole cabbage plant was obtained by cutting the stem just above the ground. The dried soil was vertically divided into three layers according to its depth (top, 0−2 cm; middle, 2−10 cm; bottom, 10−18 cm), and the root was removed from the soil. All samples were immediately weighed and stored in a freezer (below −20 °C) until analysis. In the case of the leaf treatment, the surface of treated leaves was rinsed with 100 mL of methanol per leaf. The rinsed leaf was cut into small pieces and homogenized with 20 mL of methanol at 10000 rpm and 0 °C for 10 min using a homogenizer AM-8 (Nissei Ltd., Japan). The homogenate was vacuum filtered to separate the extract and the residue. The residue remaining after filtering was extracted again in the same manner, and the filtrate was combined. The process was repeated using methanol/water (4/1, v/v). Each aliquot of the surface rinse, methanol, and methanol/water extracts was analyzed with LSC, HPLC, and 2D-TLC. The extracted residues were air-dried and individually combusted for LSC analysis. For the soil treatment, a portion of each soil layer was subjected to a combustion analysis to determine the residual amount of 14C. Approximately 50 g of the evenly mixed top layer soil for each label was transferred into 200 mL plastic centrifuge bottles, and 100 mL of methanol was added. The bottle was mechanically shaken for 10 min with a Taiyo SR-IIw recipro-shaker and then centrifuged at 5000 rpm at 4 °C for 10 min using a himac CR20G high-speed refrigerated centrifuge (Hitachi Ltd., Japan). The extract was recovered from the bottle by decantation, the residues were repeatedly extracted twice in the same manner, and then the extracts were combined. The procedure was repeated using methanol/concentrated HCl (100/1, v/v). After radioassay by LSC,
Chromatography. The reversed-phase (RP) HPLC analysis of metabolites within the leaf surface rinse and extract was conducted using a Hitachi LC module (model L-7000) equipped with a SUMIPAX ODS A-212 column (5 μm, 6 mm i.d. × 15 cm, Sumika Chemical Analysis Service (SCAS), Ltd.) at a flow rate of 1 mL min−1. The following gradient system was operated as the typical analysis with acetonitrile containing 0.05% formic acid (solvent A) and distilled water with 0.05% formic acid (solvent B): 0 min, % A/% B, 5/95; 0−3 min, 5/95, isocratic; 3−10 min, 45/55 at 10 min, linear; 10−70 min, 75/25 at 70 min, linear; 70−71 min, 5/95 at 71 min, linear; 71−80 min, 5/95, isocratic (HPLC method 1). For the separation of four isomers of 2, the gradient system as follows was applied: 0 min, % A (acetonitrile)/% B (water), 53/47; 0−3 min, 53/47, isocratic; 3−43.7 min, 58/42 at 43.7 min, linear; 43.7−44 min, 75/25 at 44 min, linear; 44−51 min, 53/47 at 51 min, linear; 51−60 min, 53/47, isocratic (HPLC method 2). The chiral analysis was conducted with a Shimadzu LC-10AT HPLC system connecting two SUMIPAX DINO2 columns (5 μm, 4 mm i.d. × 25 cm, SCAS) and one CHIRALCEL OD-H column (5 μm, 4.6 mm i.d. × 25 cm, Daicel Chemical Industries, Ltd.) in series using an isocratic eluent of nhexane/ethanol, 1000/0.5 (v/v), at a flow rate of 0.9 mL min−1. The radioactivity eluted was monitored with a Flow Scintillation Analyzer Radiomatic 500TR (Perkin-Elmer Co., Ltd.) or Ramona (Raytest, Germany) radiodetector equipped with a 500 μL liquid cell using Ultima-Flo AP (Perkin-Elmer, Co., Ltd.) as the scintillator. The detection limit of the HPLC analyses was 30 dpm. The typical retention times of 1-RTZ and 1-RTE and their related reference standards have been reported previously.2−4 One- or two-dimensional thin-layer chromatography (1D- or 2DTLC) was carried out for an analytical purpose using precoated silica gel 60F254 thin-layer chromatoplates (20 × 20 cm, 0.25 mm thickness; E. Merck). The nonradiolabeled reference standards were detected by exposing the chromatoplates to ultraviolet light or spraying bromocresol green reagent for direct visualization. Autoradiograms were prepared by transcribing the TLC plates to BAS-IIIs Fuji imaging plates (Fuji Photo Film Co., Ltd.) for several hours. The radioactivity in each spot exposed onto the imaging plate was detected by a BioImaging Analyzer Typhoon (GE Healthcare). The solvent systems for 2D-TLC were chloroform/methanol, 9/1 (v/v), and toluene/ethyl acetate/acetic acid, 5/7/1 (v/v/v). For the analysis of 2, n-hexane/ toluene/acetic acid, 3/15/2 (v/v/v) was applied for 1D-TLC development. The typical Rf values of 1-RTZ and 1-RTE and their related reference standards have been reported previously.2−4 Spectroscopy. For NMR spectrometric analyses, one-dimensional (1H, 13C, distorsionless enhancement by polarization transfer (DEPT), nuclear Overhauser effect (NOE) difference) and two-dimensional experiments (1H−1H correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple-bond connectivity (HMBC), NOE correlated spectroscopy (NOESY)) were employed in d-chloroform including tetramethylsilane (TMS) using a Varian Mercury 400 (Varian Technologies Ltd.) spectrometer (400 MHz). Liquid chromatography−electrospray ionization−mass spectrometry (LC-ESI-MS) analysis was conducted using a Waters Micromass ZQ spectrometer equipped with a Waters separation module 2695 and photo array detector 2996 as a liquid chromatograph. For the conventional analysis of metabolites, HPLC method 1 was applied with the analytical parameters controlled by MassLynx software (version 4.00) as shown: source temperature, 100 °C; desolvation temperature, 350 °C; capillary voltage, 3.2 kV; cone voltage, 10−40 V. For the analysis of 2, conditions of source temperature, 70 °C, and desolvation temperature, 300 °C, were selected to mitigate its thermal degradation and the gradient system as follows was applied: 0 min, % A (acetonitrile)/% B (methanol/20 mM ammonium acetate (20/10, v/v))/% C (water), 5/30/65; 0−50 min, 20/30/50, linear. Radioanalysis. Radioactivity in the liquid surface rinse and extract from plant was determined by mixing each aliquot with 10 mL of Packard Emulsifier Scintillator Plus and analyzed by liquid scintillation counting (LSC) with a Packard model 2900TR spectrometer. The background level of radioactivity in LSC was 30 dpm, which was 2609
dx.doi.org/10.1021/jf203903r | J. Agric. Food Chem. 2012, 60, 2607−2616
Journal of Agricultural and Food Chemistry
Article
Table 1. Distribution of Radioactivity after Foliar Applications
a
nd, not detected.
Table 2. Distribution of 1 and Its Metabolites in Treated Cabbage Leaves
nd, not detected; bConsisted of multiple components, each of which amounted to
